Comparison of retroviral-mediated gene transfer into cultured human CD34+ hematopoietic progenitor cells derived from peripheral blood, bone marrow, and fetal umbilical cord blood.
Genetic alteration of stem cells ex vivo followed by bone marrow transplantation could potentially be used in the treatment of numerous diseases and malignancies. However, there are many unanswered questions as to the best source of hematopoietic cells for long-term reengraftment and the most effective way to introduce foreign genes into this target cell. We have compared retroviral-mediated gene transfer into CD34+-enriched cells derived from peripheral blood (PB), bone marrow (BM), or fetal umbilical cord blood (CB). Cells from all three sources that had been expanded ex vivo in the presence of stem cell factor (SCF), interleukin-3 (IL-3), IL-6, and granulocyte colony-stimulating factor (G-CSF) showed transduction efficiencies ranging from 5-45%, as measured by acquisition of G418 resistance. The average efficiencies of gene transfer from multiple experiments for PB, BM, and CB were not statistically different. To determine the effect of ex vivo expansion on gene transfer into CB CD34+ cells, we compared the transduction efficiencies of cells exposed to virus immediately after harvest and CD34 selection or after 6 days of culture CD34+ CB cells were more effectively transduced after expansion in culture, showing gene transfer efficiencies 3- to 5-fold higher on day 6 compared with day 0. Last, we examined retroviral transduction via spinoculation of CB CD34+ cells and found it to be approximately as effective as our standard transduction with no significant loss of cell viability as measured by colony formation in semi-solid medium.
Campain
J A
JA
Division of Medical Oncology, University of Colorado Health Sciences Center, Denver, USA.
Terrell
K L
KL
Tomczak
J A
JA
Shpall
E J
EJ
Hami
L S
LS
Harrison
G S
GS
D000818:Animals; D001773:Blood Cells / Q000187:drug effects / Q000378:metabolism; D001854:Bone Marrow Cells / Q000187:drug effects / Q000378:metabolism; D001943:Breast Neoplasms / Q000097:blood; D002478:Cells, Cultured; D002498:Centrifugation; D003114:Colony-Forming Units Assay; D017077:Culture Media, Conditioned; D004351:Drug Resistance / Q000235:genetics; D005069:Evaluation Studies as Topic; D005260:Female; D005312:Fetal Blood / Q000166:cytology*; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics*; D005839:Gentamicins / Q000494:pharmacology; D016298:Hematopoietic Cell Growth Factors / Q000494:pharmacology; D006412:Hematopoietic Stem Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D019868:Kanamycin Kinase / Q000096:biosynthesis / Q000235:genetics*; D051379:Mice; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D012190:Retroviridae / Q000235:genetics*; D014162:Transfection / Q000379:methods
D017077:Culture Media, Conditioned; D005839:Gentamicins; D016298:Hematopoietic Cell Growth Factors; D011993:Recombinant Fusion Proteins; C010680:antibiotic G 418; D019868:Kanamycin Kinase